An Imperfect Western
After finishing the Western Blot gels last week, I was ready to begin the actual test this week. A Western Blot test usually requires about two days to complete because it must be incubated overnight.
As soon as I arrived at the lab on Monday, I began my Western Blot test. I first unpacked the gels I had previously made. The gels must then be placed in a new container that will allow for an electric current to pass through. Once the gel is set in the container, the comb can be removed and each protein sample can be loaded into the separate channels left by the comb. An electric current is them pumped through the gel for 75 minutes. For me, almost all of these first steps went smoothly. The only area that I had issues with was loading the channels with the protein samples. Rather than loading 10 microliters into each channel, I loaded 2.5 microliters. Luckily this mistake was minor and I was able to load 7.5 more microliters into each channel.
The gel is then removed from the electric current housing. Each gel is then removed and placed in a “sandwich.” These sandwiches are made up of two sponges, a gel, a membrane, and gel paper. After the gel is placed in the sandwich and has another electric current run through it overnight, the proteins will transfer from the gel to the gel paper. Once the proteins are on the gel paper, they are much easier to work with and can be scanned in an imaging machine.
On Tuesday I arrived at the lab to remove the gel paper from the sandwich and finish my first Western Blot. After I removed the gel paper, I placed them in packets of an antibody solution. The antibody will bind to the protein and allow the imaging machine to see every protein. The gel paper then goes through several different cleansing washes before it is finally ready to image.
It was an amazing feeling, knowing that I had completed my first Western Blot. The real question was: did it turn out correctly? On Thursday, we took the gel paper to the imager and scanned it. It appeared that everything turned out well except for one channel. It is possible that I forgot to load the full amount of protein sample into one of the channels. Although this did not compromise the entire test, it was a rather large mistake and I will have to do another Western Blot of the same proteins.
The picture attached to this post was taken during the first step of testing. The plastic housing holds the gel that I previously made and the blue line in the gel is the protein samples that were recently loaded into the gel. An electric current is being pushed through the gel in order to distribute the proteins through the gel based on weight.
Although my first Western Blot test was not completely successful, I now have the skills to do another test completely independently. I am excited to begin my next Western Blot test and complete every step perfectly and achieve a better final result.